Abstract
Adenine nucleotide metabolism was characterized in intact insulin secreting HIT-T15 cells during the transition from non-stimulated (i. e. 0.2 mM glucose) to the glucose-stimulated secretory state. Metabolic dynamics were monitored by assessing rates of appearance of 18O-labeled phosphoryls of endogenous nucleotides in cells incubated in medium enriched in [18O]water. Most prominent of the metabolic alterations associated with stimulated insulin secretion was the suppression in the rate of adenylate kinase (AK)-catalyzed phosphorylation of AMP by ATP. This was manifest as a graded decrease of up to 50% in the rate of appearance of beta-18O-labeled species of ADP and ATP and corresponded to the magnitude of the secretory response elicited over a range of stimulatory glucose concentrations. The only nucleotide exhibiting a significant concentration change associated with suppression of AK activity was AMP, which decreased by about 50%, irrespective of the glucose concentration. Leucine-stimulated secretion also decreased the rate of AK-catalyzed phosphotransfer. This secretory stimulus-related suppression of AK-catalyzed phosphotransfer occurs within 45 s of glucose addition, precedes insulin secretion, depends on the internalization and metabolism of glucose, and is independent of membrane depolarization and the influx of extracellular calcium. The secretory stimulus-induced decrease in AK-catalyzed phosphotransfer, therefore occurs prior to or at the time of KATP+ channel closure but it is not associated with or a consequence of events occurring subsequent to KATP+ channel closure. These results indicate that AK-catalyzed phosphotransfer may be a determinant of ATP to ADP conversion rates in the KATP+ channel microenvironment; secretory stimuli-linked decreased rates of AK-catalyzed ADP generation from ATP (and AMP) would translate into an increased probability of ATP-liganded and, therefore, closed state of the channel.
Highlights
Which increases the probability of its “closed” status [2,3,4,5,6]; when liganded with ADP the “open” state predominates and the Kϩ conductance increases [7, 8]
This secretory stimulus-related suppression of adenylate kinase (AK)- One currently held view of how glucose effects a more closed catalyzed phosphotransfer occurs within 45 s of glucose (ATP-liganded) state of this channel is through changing the addition, precedes insulin secretion, depends on the internalization and metabolism of glucose, and is independent of membrane depolarization and the influx of extracellular calcium
Detectable nor correlated with secretory stimulus-induced changes in KAϩTP channel operation, we reasoned that the dynamic transitions of the open/closed states of the channel may be related to a dynamic rather than a static characteristic of adenine nucleotide metabolism
Summary
HIT Cell Cultures—HIT cells were grown and maintained in RPMI 1640 culture media supplemented with 10% fetal bovine serum, under 5% CO2, 95% O2 at 37 °C, as described previously [16]. All studies were performed on HIT cell passages between 70 and 75 which have been previously shown to secrete insulin in response to glucose [17]. HIT cells were preincubated for 70 min at 37 °C in 15 ml of Krebs-Ringer bicarbonate (KRB) medium consisting of 118.5 mM NaCl, 2.54 mM CaCl21⁄7H2O, 1.19 mM KH2PO4, 4.74 mM KCl, 25 mM NaHCO3, 1.19 mM MgSO41⁄77H2O, 10 mM HEPES pH 7.4, 0.1% bovine albumin, and 0.2 mM glucose to lower insulin secretion to basal levels. After the 71-min preincubation the KRB medium was removed and 5 ml of KRB, enriched with a 10 – 40% atom excess of [18O]water, was added to the cells. The [18O]water-enriched KRB medium contained a predetermined glucose concentration ranging from 0.2 to 2.8 mM, along with other agents described in the figure legends.
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